Syntheses, characterization, and anti-cancer activities of pyridine-amide based compounds containing appended phenol or catechol groups

Article abstract of DOI:10.1007/s12039-014-0671-3

Several pyridine-amide compounds appended with phenol/catechol groups are synthesized. These compounds consist of protected or deprotected phenol/catechol groups and offer pyridine, amide, and phenol/catechol functional groups. All compounds have been wel

Full text of DOI:10.1007/s12039-014-0671-3

c
J. Chem. Sci. Vol. 126, No. 4, July 2014, pp. 1091–1105. ꢀ Indian Academy of Sciences.
Syntheses, characterization, and anti-cancer activities of pyridine-amide
based compounds containing appended phenol or catechol groups
AFSAR ALIa, DEEPAK BANSALa, NAGENDRA K KAUSHIKb, NEHA KAUSHIKb,
EUN HA CHOIb and RAJEEV GUPTAa,∗
aDepartment of Chemistry, University of Delhi, Delhi 110 007, India
bPlasma Bioscience Research Center, Kwangwoon University, Seoul 139 701, South Korea
e-mail: rgupta@chemistry.du.ac.in
MS received 26 December 2013; revised 26 March 2014; accepted 9 April 2014
Abstract. Several pyridine-amide compounds appended with phenol/catechol groups are synthesized. These
compounds consist of protected or deprotected phenol/catechol groups and offer pyridine, amide, and phe-
nol/catechol functional groups. All compounds have been well-characterized by various spectroscopic methods,
elemental analysis, thermal studies, and crystallography. The biological activities of all compounds were inves-
tigated while a few compounds significantly decreased the metabolic viability, growth and clonogenicity of
T98G cells in dose dependent manner. Accumulation of ROS was observed in T98G cells, which displayed a
compromised redox status as evident from increased cellular Caspase 3/7 activity and formation of micronuclei.
The in silico pharmacokinetic studies suggest that all compounds have good bioavailability, water solubility
and other drug-like parameters. A few compounds were identified as the lead molecules for future investiga-
tion due to their: (a) high activity against T98G brain, H-460 lung, and SNU-80 thyroid cancer cells; (b) low
cytotoxicity in non-malignant HEK and MRC-5 cells; (c) low toxic risks based on in silico evaluation; (d) good
theoretical oral bioavailability according to Lipinski ‘rule of five’ pharmacokinetic parameters; and (e) better
drug-likeness and drug-score values.
Keywords. Amide; phenol/catechol; Growth kinetics; T98G cell; In-silico Pharmacokinetics.
1. Introduction
significant anti-cancer and anti-microbial activities
with negligible or no toxicity against the normal cells.16
Notably, the pharmacological properties of phenol
and catechol based molecules are well-studied due
to their potent anti-oxidant properties in addition to
several other important biological roles. Phenol (or
catechol) containing copolymers are known to have
immune-stimulating and anti-tumor activities, as these
molecules adopt a rigid conformation which favours the
activation of immune-competent cells.17 Further, few
catechol derivatives are reported to have analgesic and
anaesthetic action and their activity decreases with the
size of the molecule.18
Importantly, presence of several hydroxyl groups
(phenol or catechol) in a molecule is expected to
enhance their affinity toward protein and nucleic
acid due to the presence of potential hydrogen bond
(H-bond) donors and acceptors, as has been ele-
gantly shown with synthetic systems.19–28 Interestingly,
despite potential application of both amide group as
well as phenol/catechol synthons; hybrid molecules
have largely remained unexplored. Recently, an amide
derivative containing phenol/catechol group showed
radical scavenging activities for the treatment of skin
An amide linkage, –CO–NH-, is a core structural
unit in the skeleton of proteins and pervasive in nature.
Amide-based molecules are considered to be suitable
for drug research due to their biological compatibil-
ity. Development of new amide derivatives has been
actively persuaded due to their potent application
in vast biological activities, such as fungicidal,1–4
herbicidal,5–8 insecticidal,9,10 anti-cancer,11,12 and anti-
bacterial,13 etc. In this context, derivatives of pyridine-
carboxamide are reported to show important analgesic,
anti-inflammatory and anti-pyretic activities.14 Many
diamide derivatives have also exhibited higher larvi-
cidal activity against culex mosquito which acts as
vectors for the transmission of serious diseases, such
as filariasis, West Nile virus, dengue, yellow fever,
chikungunya and other encephalitides.15 Reports
have also shown the usefulness of N,Nꢁ-dipyridyl-
2,6-pyridinedicarboxamides in treatment of atheros-
clerosis.14 We have also reported the coordination com-
plexes of several amide-based compounds displaying
∗For correspondence
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Afsar Ali et al.
ageing, similar to that of trans-resveratrol.29 This paper
reports a series of amide-based molecules appended
with phenol and catechol groups and their activities
against various cancer and non-malignant human cells.
After in vitro screening on various malignant and non-
malignant human cells, we have also shown the growth
kinetics, reactive oxygen species (ROS) generations,
clonogenicity, caspase 3/7 activity and in silico pharma-
cokinetics studies of such molecules.
study, we performed MTT assay, analysis of cell mor-
phology, growth kinetics assay, clonogenic assay, ROS
measurement and caspase 3/7 assay on cells treated by
the synthesized compounds.
2.3 Syntheses
The protected compounds H2Lp−OMe (1P ), H2Lm−OMe
(2P ) and H2Lmd (3P ) were synthesized as reported
earlier.19
2. Experimental
2.3a N-(4-methoxyphenyl)picolinamide (4P ): To a
solution of pyridine-2-carboxylic acid (2.0 g, 0.016
mmol) in pyridine (20 mL) was added 4-methoxy ani-
line (2.0 g, 0.016 mmol) and triphenylphosphite (5.2 g,
0.017 mmol) while stirring. The resulting mixture was
heated to 110◦C and stirred for 6 h. Excess pyridine
was removed under vacuum followed by the addition
of 50 mL dichloromethane. The resulting product was
extracted in 150 mL 1:1 (v/v) aqueous hydrochloric
acid. The acidic aqueous extract was neutralised by the
addition of solid sodium bicarbonate. The white solid
separated was filtered, washed thoroughly with water
and air dried. The resulting solid was recrystallized
from a saturated solution of compound in methanol-
2.1 Chemicals
All reagents were obtained from the Sigma-Aldrich and
used without further purification. Solvents were puri-
fied and dried according to the literature methods.30
The 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetra-
zoliumbromide, bis-benzimidazole derivative, N-[2-
hydroxyethyl]piperazine-N-[2-ethanesulfonic acid]
buffer, trypan blue, crystal violet dye and trypsin-EDTA
were obtained from the Sigma Chemical Co., Korea.
Dulbecco’s modified phosphate buffered saline (PBS),
Dulbecco’s modified eagle’s medium (DMEM), fetal
bovine serum (FBS) were obtained from the Hyclone,
Korea. 2,7-Dichlorofluorescin diacetate (DCFH-DA)
was purchased from Molecular probes and Caspase 3/7-
Glo assay kit was obtained from Promega (Madison
USA).
1
water. Yield: 2.5 g (75%). H NMR (400 MHz, d6-
DMSO): δ 3.73 (s, 3H, OCH3), 6.92 (d, J = 8.7 Hz, 2H,
H10 & H12), 7.80 (d, J = 8.2 Hz, 2H, H9 & H13), 7.6 (t,
J = 5.96 Hz, 1H, H3), 8.04 (t, J = 7.7 Hz, 1H, H4), 8.13
(d, J = 8.0 Hz, 1H, H5), 8.71 (d, J = 4.1 Hz, 1H, H2),
10.51 (s, 1H, NH-C=O) ppm. 13C NMR (75 MHz, d6-
DMSO): δ 113.83 (C10& C12), 121.80 (C5), 122.29 (C9
& C13), 1126.80 (C3), 131.52 (C8), 138.14 (C4), 148.44
(C2), 150.10 (C6), 155.73 (C11), 162.10 (C7), 55.22
(OCH3) ppm. Selected FT-IR bands (KBr, ν cm−1):
3356 (NH), 1683 (C=O).
2.2 Human cell culture
The T98G brain cancer, H-460 lung cancer, SNU-80
thyroid cancer, non-malignant HEK kidney and non-
malignant MRC-5 lung cells were purchased from the
Korean Cell Line Bank. These cell lines were cultured
in 75 cm2 culture flasks using DMEM supplemented
with 10% FBS, 1% non-essential amino acids, 1% glu-
tamine, 1% penicillin (100 IU/mL) and streptomycin 2.3b N-(3-methoxyphenyl)picolinamide (5P ): A
(100 mg/mL) or by the distributor instructions. All cul- similar procedure with an identical scale was followed
1
tures were maintained at 37◦C, 95% relative humidity for the synthesis of 5P . Yield: 2.0 g (67%). H NMR
and 5% CO2. Prior to each test, the cells were harvested (300 MHz, d6-DMSO): δ 3.71 (s, 3H, -OCH3), 6.65 (d,
by centrifugation at 1000 rpm for 5 min and diluted by J = 7.8 Hz, 1H, H11), 7.21 (t, J = 9.1 Hz, 1H, H12),
upto 104–105 cells/mL for experiment. Stock cultures 7.47 (d, J = 7.8 Hz, 1H, H13), 7.56 (s, 1H, H9), 7.63
were passaged every third day after centrifugation at (t, J = 7.8 Hz, 1H, H3), 8.02 (t, J = 7.8 Hz, 1H, H4),
1000 rpm for 5 min and seeding 8 × 103 cells/cm2 in tis- 8.11 (d, J = 6.8 Hz, 1H, H5), 8.69 (d, J = 5.9 Hz,
sue culture flasks to maintain the cells in the exponential 1H, H2), 10.55 (s, 1H, NH–C=O) ppm. 13C NMR
phase. All experiments were carried out on exponen- (75 MHz, d6-DMSO): δ 106.56 (C9), 110.26 (C13),
tially growing cells. The cell suspensions were seeded 112.90 (C11), 123.13 (C5), 127.53 (C3), 130.01 (C12),
into 5 mL media containing 100 mm cell culture plates 138.64 (C4), 140.05 (C8), 148.70 (C2), 150.30 (C6),
and incubated for approximately 20–24 h before the 160 (C10), 162.99 (C7), 55.55 (OCH3) ppm. Selected
experiments in order to reach confluency. In the present FT-IR bands (KBr, ν cm−1): 3336 (NH), 1684 (C=O).

Anti-cancer activities of pyridine-amide compounds
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2.3c N-(3,4-methylenedioxy benzene carbamoyl)pico- was followed for the synthesis of 3. Yield: 0.58 g
linamide (6P ): A similar procedure with an identical (57%).1H NMR (300 MHz, d6-DMSO): δ 6.80 (d, J =
scale was followed for the synthesis of 6P . Yield: 2.8 g 6.3 Hz, 2H, H12), 7.17 (d, J = 6.3 Hz, 2H, H13), 7.43
(78%). 1H NMR (300 MHz, d6-DMSO): δ 5.98 (s, 2H, (s, 2H, H9), 8.15 (t, J = 5.8 Hz, 1H, H4), 8.38 (d, J =
OCH2), 6.79 (d, J = 6.2 Hz, 1H, H12), 7.15 (d, J = 5.8 Hz, 2H, H3 & H5), 8.90 (broad s, OH), 10.8 (s, 2H,
6.2 Hz, 1H, H13), 7.53 (m, 1H, H3), 7.56 (s, 1H, H9), NH) ppm.13C NMR (75 MHz, d6-DMSO): δ 108.67
7.95 (t, J = 5.7 Hz, 1H, H4), 8.21 (d, J = 5.8 Hz, (C9), 113.05 (C12), 115.45 (C13), 125.07 (C3 & C5),
1H, H5), 8.64 (d, J = 4.3 Hz, 1H, H2), 10.16 (s, 130.04 (C8), 139.96 (C4), 142.58 (C11), 145.19 (C10),
NH) ppm.13C NMR (75 MHz, d6-DMSO): δ 101.09 149.32 (C2 & C6), 161.57 (C7) ppm. Selected FT-IR
(OCH2), 102.32 (C9), 108 (C12), 113.35 (C13), 122.33 bands (KBr, ν cm−1): 3167 (OH), 3367 (NH), 1661
(C5), 126.86 (C3), 132.76 (C8), 138.14 (C4), 143.48 (C=O).
(C11), 147.08 (C10), 148.43 (C2), 149.94 (C6), 162.21
(C7) ppm. Selected FT-IR bands (KBr, ν cm−1): 3275
2.4d N-(4-hydroxyphenyl)picolinamide (4): An iden-
(N-H), 1654, 1650 (C=O).
tical procedure as discussed for 1 was followed for
1
the synthesis of 4. Yield: 0.38 g (58%). H NMR
2.4 Deprotection of compounds 1P -6P
(300 MHz, d6-DMSO): δ 6.73 (d, J = 8.8 Hz, 2H, H10
& H12), 7.64 (d, J = 9.5 Hz, 2H, H9 & H13), 7.67 (t, 1H,
2.4a 2,6-Bis(4-hydroxy-benzene-carbamoyl)pyridine:
H4), 8.07 (t, J = 9.5 Hz,1H, H3), 8.15 (d, J = 5.8 Hz,
1H, H5), 8.71 (d, J = 5.8 Hz, 1H, H2), 10.41 (s, 1H,
NH-C=O), 4.54 (broad, OH ) ppm.13C NMR (75 MHz,
d6-DMSO): δ 139.96 (C4), 125.33 (C3 & C5), 148.97
(C2 & C6), 161.59 (C7), 129.47 (C8), 111.82 (C9 & C13),
108.10 (C10 & C12), 157.68 (C11) ppm. Selected FT-
IR bands (KBr, ν cm−1): 3244 (OH), 3299 (NH), 1638
(C=O).
(1). H2Lp−OMe (1.0 g, 0.0026 mmol) was dissolved in
40 mL of dry and degassed CH2Cl2 and BBr3 (0.8 mL,
0.0079 mmol) was added under the nitrogen atmo-
sphere at -70◦C. The resulting yellow slurry was stirred
for 8 h at room temperature. The slurry was slowly
quenched with the addition of MeOH. Evaporation of
the solvent gave an off-white solid which was further
1
recrystallized from methanol. Yield: 0.58 g (60%). H
NMR (300 MHz, d6-DMSO): δ 6.80 (d, J = 8.7 Hz,
4H, H10 & H12), 7.61 (d, J = 8.4 Hz, 4H, H9 & H13),
8.25 (t, J = 7.5 Hz, 1H, H4), 8.33 (d, J = 7.5 Hz,
2H, H3 & H5), 3.98 (broad s, OH), 10.85 (s, 2H, NH-
C=O) ppm.13C NMR (75 MHz, d6-DMSO): δ 139.96
(C4), 125.33 (C3 &C5), 148.97 (C2 & C6), 161.59
2.4e N-(3-hydroxyphenyl)picolinamide (5): An iden-
tical procedure as discussed for 1 was followed for the
synthesis of 5. Yield: 0.36 g (55%). 1HNMR (300 MHz,
d6-DMSO): δ 6.49 (d, J = 6.7 Hz, 1H, H11), 7.08
(t, J = 5.9 Hz, 1H, H12), 7.19 (d, J = 5.6 Hz, 1H,
H13), 7.42 (s, 1H, H9), 7.64 (t, J = 4.7 Hz, 1H, H3),
8.03 (t, J = 4.7 Hz, 1H, H4), 8.11 (d, J = 4.5 Hz, 1H,
H5), 8.69 (d, 1H, H2), 10.41 (s, 1H, NH–C=O) ppm.
13C NMR (75 MHz, d6-DMSO): δ 107.26 (C9), 110.97
(C11& C13), 122.42 (C5), 127.10 (C3), 129.35 (C12),
138.31 (C4), 139.35 (C8), 148.39 (C2), 149.76 (C6),
157.67 (C10), 162.18 (C7) ppm. Selected FT-IR bands
(KBr, ν cm−1): 3254 (OH), 3339 (NH), 1670 (C=O).
(C7), 129.47 (C8), 111.82 (C9 & C13), 108.10 (C10
&
C12), 157.68 (C11) ppm. Selected FT-IR bands (KBr, ν
cm−1): 3256 (OH), 3342 (NH), 1662 (C=O).
2.4b 2,6-Bis(3-hydroxy-benzene-carbamoyl)pyridine
(2): An identical procedure as discussed for 1 was
followed for the synthesis of 2. Yield: 0.54 g (55%).
1HNMR (300 MHz, d6-DMSO): δ 6.57 (d, J =
7.8 Hz, 2H, H11), 7.20 (t, J = 7.9 Hz, 2H, H13), 7.29
(t, J = 8.1 Hz, 2H, H12), 7.45 (s, 2H, H9), 8.20
(t, J = 7.6 Hz, 1H, H4), 8.36 (d, J = 7.5 Hz, 2H, H5 &
H3), 9.73 (broad s, OH), 10.92 (s, 2H, NH-C=O) ppm:
13C NMR (75 MHz, d6-DMSO): δ 148.97 (C2 & C6),
129.47 (C12), 139.96 (C8), 161.59 (C7), 125.33 (C3 &
C5), 108.10 (C9), 157.68 (C10), 111.82 (C11), 139.11
(C4), 111.53 (C13) ppm; Selected FT-IR bands (KBr, ν
cm−1): 3249 (OH), 3356 (NH), 1685,1664 (C=O).
2.4f N-(3, 4-dihydroxyphenyl)picolinamide (6):
An identical procedure as discussed for 1 was followed
for the synthesis of 6. Yield: 0.4 g (62%).1H NMR
(300 MHz, d6-DMSO): δ 6.70 (d, J = 8.4 Hz, 1H, H12),
7.05 (d, J = 8.4 Hz, 1H, H13), 7.42 (s, 1H, H9), 7.70 (t,
J = 5.2 Hz, 1H, H3), 8.11 (t, J = 7.3 Hz, 1H, H4), 8.18
(d, J = 7.5 Hz, 1H, H5), 8.64 (d, J = 8.8 Hz, 1H, H2)
ppm.13C NMR (75 MHz, d6-DMSO): δ 102.26 (C9),
104.05 (C12), 115.20 (C13), 125.51 (C5), 127.26 (C3),
2.4c 2,6-Bis(3,4-dihydroxy-benzene-carbamoyl)pyri- 129.64 (C8), 135.54 (C4), 138 (C11), 142.11 (C10),
dine (3): An identical procedure as discussed for 1 144.25 (C2), 151.05 (C6) 153.55 (C7) ppm. Selected

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Afsar Ali et al.
FT-IR bands (KBr, ν cm−1): 3144 (-OH, -NH), 1679
(C=O).
CB2 1EZ, UK; Tel.: +44-1223-336408; Fax: +44-
1223-336003; e-mail: deposit@ccdc.cam.ac.uk; web-
site: http://www.ccdc.cam.ac.uk).
2.5 Physical measurements
2.7 In vitro cell growth inhibition assay (MTT assay)
The elemental analysis data were obtained from the Ele-
mentar Analysensysteme GmbH Vario EL-III instru-
ment. The NMR measurements were done using an
Avance Bruker (300 MHz) or Jeol (400 MHz) instru-
ments. The Fourier Transform infrared (FTIR) spectra
were recorded using the Perkin Elmer FTIR 2000 spec-
trometer. The thermal analyses were performed with a
Shimadzu DTG-60 instrument in the temperature range
of 25–800◦C, at 5◦C min−1heating rate under nitrogen
atmosphere.
Cells were seeded in 96-well plates at a concentra-
tion of 1 × 104 cells/well in 200 μL of complete
media and incubated for 24 h at 37◦C in 5% CO2
atmosphere to allow for cell adhesion. Stock solutions
(3 mg/mL) of the compounds made in DMSO were
filter-sterilized and further diluted upto 11.71 μg/mL in
an incomplete media using PBS for treatment against
all 5 cell lines. Typically a 100 μL solution of com-
pound was added to a 100 μL solution of fresh
medium in wells to give final concentrations of 1500–
11.71 μg/mL. All assays were performed in three inde-
pendent sets of tests. Control group containing no
compound treatment was run in each assay. Follow-
ing 24 h exposure of cells to drug, each well was
carefully rinsed with 200 μL PBS buffer. Cytotoxic-
ity was assessed using MTT (3-[4,5-dimethylthiazol-
2-yl]-2,5-diphenyltetrazolium bromide). 20 μL MTT
solution (5 mg mL−1 dd H2O) along with 200 μL of
fresh complete media were added to each well and
plates were incubated for 3 h. Following incubation,
the medium was removed and the purple formazan pre-
cipitate in each well was sterilized in 100 μL DMSO.
Absorbance was measured using Biotek Synergy HT
microplate reader at 540 nm and percentage (%) viabil-
ity was calculated36–38 which was directly proportional
to metabolic active cell number as:
2.6 Crystallography
The crystals for compounds 1, 2, 4, 5 and 6 were
grown from methanol either by slow evaporation or
by vapour diffusion of diethyl ether. The x-ray diffrac-
tion data for 1, 2, 5 and 6 were collected on an
Oxford XCalibur CCD diffractometer whereas for 4
on Bruker Kappa Apex-CCD diffractometer equipped
with graphite monochromatic Mo/Kα radition (λ =
0.71073 Å).31,32 The structures were solved by the
direct methods and refined by full-matrix least-squares
refinement techniques on F2 using SHLEXL-97 in
WinGX module.33,34 The non-hydrogen atoms were
refined anisotropically. All hydrogen atoms were fixed
geometrically with Uiso values of 1.2 and 1.5 times
the Uiso values of phenylene carbon atom and pheno-
lic hydrogen atoms, respectively. For compound 5 there
was scattered electron density around the bromine atom
that could not be assigned and resulted into large R
value. There were positionally disordered oxygen atoms
O4 and O2 for compounds 5 and 6, respectively. These
atoms could be located easily and were refined with
site occupancy factor (SOF) of 0.5. This generates com-
pounds 5 and 6 with one of its oxygen atom (O4 and O2,
respectively) being disordered over two positions with
50% site occupancy. In general, all molecules produced
very thin plate-like crystals which diffracted poorly.
Due to such a problem, the final R value was quite on
the higher side. Detail of the X-ray data collection and
structure solution parameters is provided in table 1.
Crystallographic data (without structure factors) for
the structure reported in this paper have been deposited
with the Cambridge Crystallographic Data Centre
(CCDC) as supplementary publication no. CCDC-
%Viability=OD in sample well/OD in control well×100.
Results expressed as inhibitory concentration 50 (IC50)
were obtained by fitting values to trend dose-response
curves using the routines provided in Microsoft Excel.
2.8 Growth kinetics
T98G cells were seeded at 10000 cells/cm2on the cell
culture plate and their proliferation kinetics were stud-
ied at different incubation times (24, 48 and 72 h)
after compounds treatment, following trypsinization
and counting the total cells per plate microscopically
using a trypan blue dye and hemocytometer (N7-8).35–38
2.9 Clonogenic assay
942968-942972. Copies of the data can be obtained free Clonogenic assay or colony formation assay is an in
of charge from the CCDC (12 Union Road, Cambridge vitro cell survival assay based on the ability of a single

Anti-cancer activities of pyridine-amide compounds
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Afsar Ali et al.
cell to grow into a colony. The colony is defined to con- shaker at 300–500 rpm for 30 seconds. The plate was
sist of at least 50 cells. The assay essentially tests every then incubated at room temperature for 2 hours. The
cell in the population for its ability to undergo ‘unlim- luminescence of each sample was measured in a plate-
ited’ division.35–38 Clonogenic assay is the method of reading luminometer. The experiments were performed
choice to determine cell reproductive death after treat- in triplicate and repeated on two separately-initiated
ment with ionizing radiation, but it can also be used cultures.
to determine the effectiveness of drug molecules. Only
a fraction of seeded cells retains the capacity to pro-
duce colonies before or after treatment, cells will be
2.12 Micronucleus assay
seeded out in appropriate dilutions to form colonies in
The in vitro micronucleus (MN) assay is an attrac-
1–3 weeks. After harvesting with 0.05% trypsin, 150–
tive tool for measuring genotoxicity. For measuring
400 (depending on the treatment) cells were plated 10–
micronucleus, T98G cells were treated by compound
14 h before treatment in DMEM at 37◦C. Cultured
under similar conditions as used for growth kinetics and
cells were treated with doses 20 to 100 ug/mL of com-
clonogenic assay. After treatment, cell cultures were
pounds. After the treatment, cells were incubated in
grown for 24 h to allow chromosomal damage to lead
dark under humidified, 5% CO2 atmosphere at 37◦C for
to the formation of micronuclei in bi- or multinucleated
8–10 days to allow colony formation. Colonies were
interphase cells. Air-dried slides containing acetic acid–
fixed with methanol and stained with 1% crystal violet.
methanol (1:3 V/V) fixed treated cells were stained with
Colonies of more than 50 cells were counted and the
Hoechst-33342 (10 μg/mL in 0.1 M PBS), disodium
surviving fraction (SF) was calculated. Clonogenic sur-
phosphate (0.45 M) buffer containing 0.05% Tween-
vival curves were constructed from three independent
20 detergent. Slides were examined under the fluores-
experiments by least-squares regression fitting averaged
cence microscope using an UV excitation filter. Fluo-
survival levels.
rescent nuclei were visualized using a blue emission
filter. Cells containing micronuclei were counted from
>1000 cells. The fraction of cells containing micronu-
clei, called the M-fraction (%) or MN frequency was
calculated as follows:
2.10 Intracellular ROS measurement
The fluorochrome probe DCFH-DA was used to mea-
sure ROS. The non-fluorescent compound DCFH-DA
penetrates into the cell and is cleaved by intracellular
esterase, resulting in the formation of DCFH, the oxida-
tion of which (due to oxidative stress) generates the flu-
orescent compound DCF. Thus, the DCF fluorescence
represents the rate and quantity of ROS produced. After
treatment with compounds, T98G cells were incubated
in fresh media with 10 μM DCFH-DA at 37◦C for 30
minutes and washed twice with PBS and the fluores-
cence measured using plate reader with excitation at
485 nm and emission at 528 nm. Average ROS produc-
tion (relative to level of untreated controls) was calcu-
lated from four individual wells in at least three inde-
pendent plates. Mean fluorescence intensity (MFI) was
calculated after correction for autofluorescence.
M − fraction (%) = Nm/Nt × 100
where Nm is the number of cells with micronuclei and
Nt is the total number of cells analyzed. Since, micronu-
clei formation is linked to cell proliferation, micronu-
clei frequencies were normalized with respect to the cell
numbers.
2.13 In silico pharmacokinetic screening
To evaluate pharmacokinetic profile descriptors such as
cLog P (octanol/water partition coefficient) and Log S
(water solubility) were calculated using the Osiris Prop-
erty Explorer on-line system.39 The molecules were
submitted to in silico ADMET (absorption, distribution,
metabolism, excretion, and toxicity) screening using
the Osiris program. Values of drug-likeness are based
on the occurrence frequency of each fragment of the
2.11 Caspase activity assay
Following treatments T98G cells were subjected to Cas- molecule in commercial drugs while the drug-score
pase 3/7 activity measurement with Caspase-Glo assay evaluates the molecules potential to qualify for a drug
kit after 24 hour of incubation. Briefly, the plates con- and is related to topological descriptors, fingerprints
taining cells were removed from the incubator and of molecular drug-likeness, structural keys and other
allowed to equilibrate to room temperature for 30 min- properties such as cLog P and molecular mass.40,41 in
utes. 100 μl of Caspase-Glo reagent was added to each silico theoretical safety analysis was also evaluated by
well, the content of well was gently mixed with a plate Osiris software, score 1 means drug is safe and score

Anti-cancer activities of pyridine-amide compounds
<1 means drug molecule is theoretically toxic for the
1097
use. The pharmacokinetic profile, important for a good
oral bioavailability of a molecule, was also evaluated
according to the Lipinski’s ‘rule-of-five’ by using cat-
alyst and chemaxon software, which analyses features
that a drug should present to allow the absorption and
permeation across the membranes and states molecu-
lar weight <500 daltons (Da), calculated octanol/water
partition coefficient (cLog P) <5, number of hydrogen-
bond acceptors (nHba) <10, and number of hydrogen-
bond donors (nHbd) <5, as well as a fifth rule added
later, which infers the number of rotatable bonds <10.
2.14 Statistical analysis
Data is expressed as a mean SD. Statistical analysis
was performed by student student’s t-test. Results were
considered significant when P < 0.05.
Scheme 1. Synthetic procedure adopted for the protected,
1P -6P and deprotected compounds, 1-6.
3. Results and Discussion
3.1 Chemistry
νN−H and νC=O stretches for the deprotected compounds,
This work utilizes a series of pyridine-amide based 1-6, were observed in the range of 3300–3367 cm−1 and
compounds appended with either phenol or cate- 1662–1679 cm−1, respectively (table S1; supporting
chol groups for their pharmacological properties.42 information). The deprotected compounds show red–
These compounds are categorised in two classes: class shifted νC=O stretching frequencies than their protected
one consists of bis-amide compounds 1P -3P and 1-3; analogues. In addition, the resultant phenolic groups
whereas class two constitutes mono-amide analogues now show νO−H in the range of 3144-3253 cm−1 which
4P -6P and 4-6. The protected compounds 1P -6P were is significantly red-shifted than the gas-phase phenol
synthesised by the coupling of respective pyridine- suggesting possible H-bonding in all molecules. All
carboxylic acid with the required amine (3-methoxy protected as well as deprotected compounds were thor-
1
aniline, 4-methoxy aniline or 3,4-methylenedioxy ani- oughly characterized by the H and 13C NMR spec-
line) using P(OPh)3. These protected compounds on troscopy (figures S1-S12; tables S2 and S3; support-
treatment with boron tribromide provided the desired ing information). The 1H NMR spectra of all protected
compounds with free phenol or catechol groups compounds display features for –NH; Hpyridine; Harene
;
(scheme 1). It is important to mention here that some of -OCH3 or –OCH2O– groups. After deprotection with
these molecules were found to firmly chelate transition BBr3, the resonances for –OCH3 or –OCH2O– groups
metal ions;19 and therefore, likely to have strong poten- disappeared and the remaining ring protons were found
tial to interact with biologically relevant metal ions. to appear slightly up-field. In addition, phenol or cate-
In addition, the presence of amide and phenol/catechol chol protons were observed as broad resonances which
groups in the resultant coordination complexes were disappeared on deuterium exchange.
demonstrated to exhibit strong intra as well as inter-
To gain insight about the solid-state conformation,
molecular hydrogen bonding (H – bonding)19 and such all compounds were characterized by the single crys-
features depict their importance to adhere to cell mem- tal diffraction studies (figure 1). Out of six deprotected
brane and/or other biological surfaces.
molecules, two compounds 5 and 6 could only be crys-
All protected and deprotected compounds were tallized as their HBr salt with the aid of one equiv-
characterized by various spectroscopic methods. The alent of HBr thus resulting in the formation of pyri-
infrared spectra of compounds 1P -6P show νN−H and dinium ion. The compounds 1 and 2 show the pres-
νC=O stretching frequencies in the range of 3274– ence of strong hydrogen bond (H – bond) between
3356 cm−1 and 1654–1684 cm−1, respectively. The both N–H groups to that of central Npyridyl atom. Such

1098
Afsar Ali et al.
O1
O2
N2
N1
N3
O4
O3
(1)
(2)
Br
Br-
N1
N1
N2
O1
N2
O1
O2
O3
O2
(4)
(5)
(6)
Figure 1. Molecular structures of compounds 1, 2, 4, 5 and 6 with partial
numbering scheme (Thermal ellipsoids are drawn at 50% probability level
whereas only phenolic and amidic hydrogen atoms are shown for clarity).
N-H. . .Npyridyl interactions resulted in the stabilization 275◦C, respectively. In contrast, temperature of melting
of a single confirmation where two appended pheno- (Tm) for mono-amide molecules 4, 5 and 6 was found
lic rings are geometrically locked. For both 1 and 2, to be 140, 167 and 135◦C, respectively (figure S13).
the Namide . . .Npyridyl heteroatom separation was between Therefore, thermal studies display lower thermal stabil-
2.630 – 2.725 Å; suggesting strong H – bond. For ity of mono-amide compounds than that of bis-amide
mono-amide compounds, two arene rings are almost analogues.
co-planar to each other as revealed by a small angle
(2.66◦ for 5; 3.70◦ for 6; and 7.33◦ for 4). A some-
what similar situation was observed for compound 2
3.2 Pharmacology
as the aromatic rings were marginally out-of-plane (ca.
6.0◦ and ca. 9.4◦). In contrast, the para-phenolic rings
3.2a Inhibitory effects of compounds on cancer and
were significantly out-of-plane with respect to the cen-
tral pyridine ring for bis-amide compound 1 (angles
being ca. 15◦ and ca. 70◦). Notably, all these molecules
are equipped with H-bond donor –OH groups and H-
bond acceptors Oamide groups and thus self-assemble
into interesting pattern-based networks via intermolec-
ular H – bonds. Compound 3 was also characterized
crystallographically and showed the proposed struc-
ture. However, poor crystal quality and severe disor-
ders did not allow accurate structure solution and there-
fore the molecular structure is displayed in figure S13
(supplementary information).
To understand the thermal stability and decompo-
sition profile, the thermogravimetric analysis (TGA)
were performed on all six deprotected compounds
(figure S14). TGA plots for three bis-amide compounds
(1-3) show 5-8% weight loss in the temperature range
of 30–200◦C. Such a weight loss corresponded to
the release of lattice solvent molecules. TGA analysis
revealed that deprotected bis-amide compounds 1-3 are
thermally stable. In particular, the temperature of melt-
ing (Tm) for 1, 2 and 3 was found to be 240, 302 and
normal cells: The IC50 values of all compounds on the
T98G brain cancer, H-460 lung cancer, SNU-80 thyroid
cancer, non-malignant HEK kidney and non-malignant
MRC-5 lung cells were evaluated and the results are
presented in table 2. The MTT cell proliferation assay
has been widely accepted as a reliable method to mea-
sure the cell proliferation rate and conversely when
metabolic events lead to apoptosis or necrosis. The data
obtained by the MTT assay show that all compounds
have inhibitory effects on the growth of cancer cells in
a dose-dependent manner. MTT was reduced by viable
cells to coloured formazan via metabolic dehydroge-
nase enzyme and has been used for chemo and radio-
sensitivity test. All compounds inhibited the growth of
T98G, H-460, SNU-80, HEK and MRC-5 cells, with
their IC50 values ranging from 20.55 to 187.35 μg/mL
(52.9 to 873.8 μM). However 3P , 2, 3 and 6 are highly
active (IC50< 100 μM) against brain cancer cell line
and showed very less cytotoxicity on non-malignant
HEK and MRC-5 cell lines, therefore we selected these
four compounds for further experiments on brain cancer
cells (table 2).

Anti-cancer activities of pyridine-amide compounds
1099
Table 2. IC50 values (μg/mL, μM) evaluated from MTT assay on H-460 (lung cancer), T98G (brain cancer), SNU-80
(thyroid cancer), HEK (non-malignant embryonic kidney) and MRC-5 (non-malignant lung fibroblast) cells treated with
compounds 1P -6P and 1-6 for 24 h. Results are given as Mean ( SD), n = 3. Negative control cell group were treated with
medium only and positive control cell population is treated with anticancer drug actinomycin D (Act-D).
S. No.
IC50 μg/mL ( SD), μM
Malignant Cell Lines
H-460
Non-malignant cell Lines
HEK MRC-5
T98G
SNU
1P
2P
3P
4P
5P
6P
1
2
3
4
5
37.93( 5.5), 100.6
35.76( 6.7), 94.6
32.42( 6.2), 80
63.16( 4.4), 167.3
70.72( 4.9), 187.5
59.27( 3.0), 149.34 37.49( 4.9), 92.5
43.73( 4.6), 115.9
39.04( 2.8), 103.5
118.37( 9.0), 313.7 89.54( 13.4), 237.4
153.18( 10.1), 406.3 92.39( 11.5), 245
68.74( 8.9), 169.6
139.44( 31.0), 344.1
111.03( 14.0), 486.8 145.21( 13.1), 636.8 123.00( 7.9), 539.4 152.87( 16.7), 670.1 152.46( 16.2), 668.4
112.21( 15.2) 492.1 82.41 ( 6.3), 361.4
103.05( 45.1), 425.8 69.49( 6.7), 287.1
45.41 ( 13.3), 130
23.06( 2.9), 66.07
20.55( 3.6), 52.9
51.92 ( 14.2), 242.5 70.89( 3.7), 331.2
50.86( 1.6), 237.6
24.5( 4.9), 106.5
105.58( 6.2), 462.7 166.30( 14.5), 729.3 117.22( 11.6), 514.1
78.52( 12.0 ), 324.3 183.85( 16.7), 759.5 125.18( 7.3), 516.9
67.73( 6.6), 194.06 116.57( 6.4), 333.8 96.92( 10.6), 277.0
33.13( 5.3), 94.92
47.91( 3.7), 137.27 39.01( 4.2), 111.7
62.38( 2.3), 163.7
118.25( 11.6), 338.8 62.96( 6.2), 180.4
73.97( 5.6), 194.1 61.84( 1.5 ), 162.3
175.55( 19.1), 820.3 123.53( 10.3), 577.1
187.35( 25.9), 873.8 185.81( 17.3), 864.4
21.30( 3.8), 55.9
56.63( 9.3), 264.6
58.93( 6.9), 275.2
85.11( 8.7), 397.7
69.50( 10.5), 302.1 31.97( 2.8), 139
– 25.53( 7.1), 20.34
6
92.473( 10.5), 402
15.34( 5.9), 12.22
99.38( 10.4), 432
–
Act-D 29.3( 6.1), 23.34
– Not tested
Importantly, out of twelve samples, compounds 3P ,2,
3 and 6 were found to inhibit 50% T98G brain cancer
cell growth in the range from 20.55 to 32.42 μg/mL
after 24 h of treatment. Compounds 2, 3 and 6 showed
potent inhibitory activity as compared to positive con-
trol group treated with actinomycin D (Act-D) on brain
cancer cells. However, Act-D itself is highly toxic on
normal HEK cells. The results show that the present
compounds were less cytotoxic to non-malignant nor-
mal HEK and MRC-5 cells at moderate concentrations
(187.5–11.71 μg/mL) (table 2). The IC50 values of com-
pounds for MRC-5 cells were in the range from 61.84
to 185.81 μg/mL. The results indicate that 3P ,2, 3 and
6 inhibit T98G cancer cell growth which may be due
to either growth arrest or cell death. Overall, these
results clearly demonstrate that the compounds 3P ,2, 3
and 6 effectively sensitize T98G cells in a dose depen-
dent manner, which may be by increasing the mitotic
(linked to cytogenetic damage) or interphase (apop-
tosis) cell death. We further performed growth kinet-
ics assay, clonogenic assay, ROS measurement assay
and Caspase 3/7 activity in treated T98G cells to con-
firm growth inhibition, clonogenic inhibition, oxidative
stress and apoptosis, respectively.
of compounds and control drug, Act-D. Cell suspen-
sions were made in media layers of 4 mm thickness
in 6 well plates. Notably, treatment of cells with com-
pounds 3P ,2, 6 and Act-D showed less effect than those
exposed to compound 3 after 24 h of treatment. The
maximum effect was seen after exposure to compounds
3 inhibiting the cell growth from 47 to 93% at 24 h
at all concentrations. Whereas in case of compound 6,
exposure on T98G cells led to 45–87% cell death and
their viability was 12.47 and 55.68% at 24 and 72 h,
respectively. However, compound 6 showed the incuba-
tion time dependent effect on the growth of T98G cells
and having maximum inhibitory effect at 72 h of incu-
bation when compared with Act-D and other cases. In
essence, compounds 3P ,2, 3 and 6 inhibited the growth
of T98G cells in a dose and incubation dependent man-
ner. Growth pattern and density of T98g cells were also
affected after treatment (figure 3). These experiments
indicate that the present compounds inhibit T98G can-
cer cell growth which may be due to either growth arrest
or cell death.
3.2c Clonogenic Assay: Figure 4 shows the effect of
selected compounds on the colony forming capacity of
T98G cells. The clonogenic assay was employed to con-
3.2b Growth Kinetics: Growth kinetics of the T98G firm the colony formation inhibition due to treatment.
cells is shown in figure 2. Data obtained by growth Only a fraction of seeded cells retains the capacity to
kinetics assay show that the compounds 3P ,2, 3 and 6 produce colonies after cytotoxic drug treatment. Clono-
have an inhibitory effect on T98G cells that is depen- genic assay or colony formation assay is an in-vitro
dent on dose and incubation time. The culture cells assay based on the ability of a single cell to grow into
were treated at 11.71 to 187.50 μg/mL concentration a colony. This in-vitro clonogenicity method has been

1100
Afsar Ali et al.
Figure 2. Growth kinetics of T98G cells at 24, 48 and 72 h after treatment with compounds
3P , 2, 3 and 6 at 11.71–187.5 μg/mL concentrations. Untreated cells were taken as control
and all values are given as mean ( SD) of three independent experiments, n = 3.
Figure 3. The bright field images of T98G cells at 1 day after treatment with 5 and
25 μg/mL of compound 2, 3 and 6. Control cell group were treated with medium only
and positive control cell population is treated with 5 μg/mL anticancer drug actinomycin D
(Act-D). All scale bars are 100 μm.

Anti-cancer activities of pyridine-amide compounds
1101
treatment with T98G brain cells is shown in figure 5.
The decrease in MTT formazen due to growth inhi-
bition and increase in clonogenic inhibition might be
induced by increased oxidative stress that would com-
promise the survival leading to cell death in T98G cells.
Therefore to determine if treatment enhanced steady
state of oxidative stress in malignant cells, cells were
labelled with the oxidation sensitive probe (DCFDA)
that is capable of being oxidized to fluorescent product
(DCF) by ROS. The fluorescence was measured using
plate reader with excitation at 485 nm and emission at
528 nm. Mean fluorescence intensity (MFI) was calcu-
lated after correction for auto-fluorescence. We found
that the compounds 3P, 2, 3, 6 and Act-D enhanced ROS
production at 24 h inside the T98G cells. Treatment by
compounds 3P, 2, 3, 6 and Act-D increases the DCF flu-
orescence by 232.1% (MFI-162.5), 188.5% (MFI-132),
212.8% (MFI-149), 239.2% (MFI-167.5) and 241%
(168.75), respectively at concentration 2.92 μg/mL in
T98G cells as compared to untreated control (MFI-70).
However, more significant change in ROS production
was observed in cells treated with 1.46 μg/mL concen-
tration of compounds when compared with Act-D (pos-
itive control). Interestingly, liquid holding conditions
had no influence on oxidative stress in any of the cell
line studied, as the DCF fluorescence of untreated cells
was similar in PBS and growth media (MFI in PBS,
DCF = 75; MFI in growth media, DCF = 70).
3P
Figure 4. Effect of compounds 3P , 2, 3 and 6 on colony
forming capacity or clonogenic survival of exponentially
growing T98G cell lines studied by clonogenic assay at
11.71–93.75 μg/mL concentrations. Data presented are mean
values from three independent observations, n = 3.
recognized as a valid surrogate assay for tumor growth
in vivo. It was observed as the compound concentra-
tion increases; the treatment enhances cell death and
inhibits the colony formation capability of the T98G
cell population. Clonogenic assay shows the effect of
compounds 3P ,2, 3 and 6 on the colony forming capac-
ity and survival of exponentially growing T98G cells.
It was seen that the survival fraction of T98G cells was
drastically decreased after treatment with compounds
(figure 4). Importantly, treatment with compounds 3P ,2,
3 and 6 enhances the cell death and also inhibits colony
formation capability in the T98G cell population in a
concentration dependent manner. Survival fraction of
T98G cells declined on treatments with different con-
centration (93.75-23.43 μg/mL) which is evident by the
reduction in the number of colonies formation. Even
low dose (23.43 μg/mL) of compounds 3P ,2, 3 and
6 showed significant decline in the % colony survival
which was 33.33, 60.0, 29.66, and 73.33, respectively.
A drastic decline in the % colony survival was observed
on exposure to higher concentration (46.87 μg/mL) of
compounds 3P ,2, 3 and 6. Notably, compounds 2 and 6
exhibit higher inhibitory effect on colony formation of
T98G cells, at all doses. Compound 6 shows maximum
inhibition at % colony survival 29.66, 13.33 and 0 on
exposure with 23, 46 and 93 μg/mL of doses, respec-
tively. Such outcome reflects the significant inhibitory
effect on colony formation capabilities of brain cancer
cells at all dosages.
3.2e Caspase 3/7 activity: To assess the effect of
compounds on the activity of Caspase 3/7 in brain can-
cer cells, the human T98G cell line were treated with
different doses of compounds. It is widely assumed that
Figure 5. Effect of compounds 3P , 2, 3 and 6 on the reac-
tive oxygen species (ROS) level in T98G cell lines. ROS
values (in MFI, mean florescence intensity) presented are
mean SD (n = 3).
3.2d ROS detection in cells: Reactive oxygen spe-
cies (ROS) measurement of selected compounds on

1102
Afsar Ali et al.
the apoptotic death of mammalian cells is closely asso- After treatment by compounds, cell cultures were
ciated with the activation of Caspases. Caspase-3 and -7 grown for 24 h to allow chromosomal damage to lead
play a crucial role as early apoptosis biochemical mark- to the formation of micronuclei in bi- or multinucle-
R
ers in mammalian cells. Caspase-Gloꢀ 3/7 assay uses ated interphase cells. Figure 7 shows micronucleus fre-
a luminogenic substrate containing the DEVD sequence quency at 24 h for T98G brain cancer cells after treat-
which is selective for caspase-3 and -7. The caspases ment by 5–25 μg/mL concentration of compound 2, 3
activity in both non-treated and treated cancer cells and 6. The frequency of untreated cells with micronu-
were measured after 24 h and the result are shown in clei has been in the range of 2–4%. Notably, treatment
figure 6.
with 5 μg/mL of 2, 3 and 6 exposure induce a significant
Caspase activity level detected in non-treated con- level of micronuclei formation in T98G cells, and the
trol cells corresponded to the portion of apoptotic cells range of micronucleus is 7%, 9% and 17%, respectively.
present in the naturally growing population due to nat- The micronuclei frequency increased from 4% (con-
ural aging. In treated cells, activities of Caspase-3 and trol) to 12%, 19% and 25% for 15 μg/mL concentration
-7 increased from 1.3-2.0 fold (P < 0.05) over basal lev- treated cells with 2, 3 and 6, respectively, at 24 h cul-
els signifying an activation of these caspases in T98G ture. However, 25 μg/mL concentrations of 2, 3 and 6
cancer cells treated with the compounds. Notably, treat- further increases micronuclei frequency from 4% (con-
ment with 3 and 6 shows more than two-fold increase trol) to 15%, 26% and 33%, respectively. The micronu-
in Caspase activity when compared with untreated con- clei in T98G cells is formed by DNA strand breaks gen-
trol. In general, the results show that these compounds erated during the faulty excision repair process due to
are inducing apoptosis in T98G cell population in dose compound genotoxicity. The remaining unsealed DNA
dependent manner at 24 h after treatment.
leads to the formation of micronuclei in subsequent
mitosis, and cells with micronuclei are found to be
associated with loss of reproductive capacity.
3.2f Genotoxicity against brain cancer cells: In
recent years, the in-vitro micronucleus (MN) assay has
become an attractive tool for measuring genotoxicity
by any physical and chemical agents, because of its
capacity to detect clastogenic and aneugenic events,
simplicity of scoring, accuracy, multipotentiality, and
wide applicability in different cell type. For measuring
micronucleus, T98G cells were treated with compound
2, 3 and 6 under similar conditions and concentrations
as were used for growth kinetics and clonogenic assay.
3.2g In silico pharmacokinetics: A probable drug
molecule, besides having good biological activity, must
have good pharmacokinetic accessibility in biologi-
cal system. To access the pharmacokinetic profile of
synthesized molecules, well-validated in-silico tools:
Osiris, Chemaxon and Catalyst were used. These tools
have been validated with over 7000 drug molecules
available in the market. The analysis of theoretical toxi-
city risks for the synthesized compounds using OSIRIS
program shows that compounds 3P , 6P and 1-6 are
not toxic (score for mutagenicity, tumorigenicity, irri-
tating and reproductive effect is 1) and can be used
as the therapeutic molecules (figure 8). On the other
side, compounds 1P , 2P , 4P and 5P were found to have
some toxicity effect. In particular, compounds 1P and
4P showed high mutagenic effects (score 0.6) while 2P
and 5P showed high reproductive effects (score 0.6).
As these compounds were considered for oral delivery,
they were submitted to the analysis of Lipinski ‘rule
of five’, drug-likeness and drug score by using catalyst
software (table 3). The results indicate that all effec-
tive molecules 3P ,2, 3 and 6 fulfil rule and their drug-
likeness property, such as molecular weight (230-405),
Log P (1.49-3.48), nHBA (5-9), nHBD (2-6) and num-
ber of rotatable bonds (rotb) (2-4) were similar to the
commercial drugs (table 3).
3P
Figure 6. Compounds 3P , 2, 3 and 6 induced activation of
caspase 3/7 in T98G cell line. Apoptosis was assessed by
measuring active caspase-3/7 using the Promega Caspase-
3/7 assay kit. Experiments were performed in triplicate and
repeated two times.
Finally, we evaluated all molecules as the potential
drugs by calculating their drug-likeness and drug-score.

Anti-cancer activities of pyridine-amide compounds
1103
Figure 7. The various treated cells that observed in the micronucleus (MN)
assay at concentration 5, 15 and 25 μg/ml of compound 2, 3 and 6. Control cell
groups were treated with medium only and positive control cell population is
treated with 5 μg/mL anticancer drug actinomycin D (Act-D). The cells shown
with one or many micronucleus were scored for MN. All scale bars are 100 μm.
Drug-likeness is related to the similarity with the com- score for the commercial drug, Act-D was 0.42. Impor-
mercial drugs (-2.75-1.74). The drug score for all com- tantly, drug score for the commercial drug, Act-D is
pounds were in the range of 0.27–0.87 whereas the drug less than that of our synthesized compounds. Among
all molecules, compounds 1-6 showed the best values
of drug-score (0.68–0.87) with no toxicity risk, which
point towards their further exploration.
It is well-established that subtle structural changes
in an organic compound considerably influences its
biological activity. A few illustrative examples are
discussed here. Shin and co-workers43 have reported
the anti-amyoidogenic activity of a large number of
molecules where a substituted phenol ring is part of
the framework. Notably, the position and number of the
hydroxyl group onto the arene ring was considered to
be an important design criterion for the observed activ-
ities. Borges and co-workers44 have evaluated the anal-
gesic properties of several hybrid amide-based com-
pounds containing phenol rings where the position of
the hydroxyl group onto the arene ring has been var-
Figure 8. In silico drug safety analysis by Osiris software.
ied and was found to significantly affect the pharmaco-
logical properties. Kitamura and co-workers45 have
synthesized a few stilbene derivatives where subtle
High score is considered as indicator for safe drugs (Safe
drug: Score 1; Medium Risk: Score 0.9–0.7; High Risk:
<0.7).

1104
Afsar Ali et al.
Table 3. Pharmacokinetic parameters (Catalyst, Chemaxon and Osiris softwares).
Compounds
nHba
nHbd
nrotb
MW
cLog P
Drug-likeness
Drug Score
1P
2P
3P
4P
5P
6P
1
2
3
4
5
7
7
9
4
4
5
7
7
9
4
4
5
28
2
2
2
1
1
1
4
4
6
2
2
3
6
6
6
4
3
3
2
4
4
4
2
2
2
8
377
377
405
228
228
242
349
349
381
214
214
230
1255
3.07
3.07
3.48
1.99
1.99
2.19
2.68
2.68
2.08
1.79
1.79
1.49
0.24
0.05
0.81
-2.75
0.26
1.05
-2.54
1.55
1.06
0.45
1.74
1.27
0.64
7.09
0.27
0.30
0.37
0.35
0.39
0.48
0.75
0.71
0.68
0.87
0.84
0.79
0.42
6
Act-D
structural changes were noted to control the estro- and 6 presented the overall best parameters including:
genic activities; in particular, para-hydroxyl group (a) high activity against T98G brain cancer cell line;
on the first phenyl ring and peripheral substituent(s) (b) low cytotoxicity on HEK and MRC-5 cells at effec-
on the second phenyl ring were considered impor- tive concentrations; (c) low in-silico toxicity risks; (d)
tant structural parameters. Zhou and co-workers46 have good oral bioavailability according to the Lipinski ‘rule
synthesized synthetic analogues of resveratrol and of five’; and (e) better drug-likeness and drug-score
found that the molecules bearing ortho-dihydroxyl values, nearly similar or better than the commercial
or 4-hydroxy-3-methoxyl groups possess significantly drugs. Based on MTT, growth kinetics, clonogenic and
higher antioxidant activity than those bearing no such ROS measurement assays, it is concluded that treatment
functionalities. These examples adequately illustrate has significant inhibitory effect on T98G cell growth
the importance of designed organic molecules con- and created oxidative stress in the cells. Production
taining hybrid functional groups and further show of ROS is initiated immediately after treatment with
that the position as well as the number of hydroxyl these compounds, followed by micronuclei formation
groups on arene ring can influence the biological acti- and apoptotic cell death, which is confirmed by MN
vities. Interestingly, out of twelve compounds that have and Caspase 3/7 assay. The results suggest that apop-
been assayed; compounds containing catechol rings (3 tosis occurred in the cells treated with compounds as
and 6) displayed high anti-cancer activity. It is therefore the level of caspases was increased manifold over the
suggested that not only type of functional group(s) but basal level of untreated T98G cells. This could be due
also their number and position possibly influence the to enhanced amount of ROS inside cells, which has
biological activity of a potential drug molecule.
been shown to induce apoptosis in several human can-
cer cells. Decreased viability of T98G cells strongly
suggests that the oxidative stress induced by these com-
pounds resulted in the observed cell death or apopto-
sis. We conclude that the oxidative stress induced by
the present compounds leads to sensitization in T98G
malignant cell line due to the accumulation of ROS
and compromised redox status. The importance of such
work lies in the possibility that the next-generation
molecules might be more efficacious as the anticancer
agents and our future work is directed towards such a goal.
4. Conclusions
Several biologically significant pyridine-amide based
compounds were synthesised, characterized and their
medicinal applications were investigated. The results
of treatment against human T98G brain cancer, H-460
lung cancer and SNU-80 thyroid cancer cells were
compared with the non-malignant HEK and MRC-5
cells. All compounds inhibited the growth of T98G, H-
460, SNU-80, HEK and MRC-5 cells with their IC50
values ranging from 20.55 to 187.35 μg/mL (52.9 to
873.8 μM); however 3P , 2, 3 and 6 were particularly
Supplementary Information
active (IC50< 100 μM) against brain cancer cells and Figures for NMR spectra, TG analysis and X-ray struc-
showed very less cytotoxicity on non-malignant HEK ture, and tables for FTIR and NMR spectra data can be
and MRC-5 cells. In particular, compounds 3P , 2, 3 seen at www.ias.ac.in/chemsci.

Anti-cancer activities of pyridine-amide compounds
1105
20. Kumar G, Aggarwal H and Gupta R 2013 Cryst. Growth
Des. 13 74
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RG acknowledges the Department of Science and Tech-
nology (DST) for financial support and CIF-USIC of
this university for the instrumental facilities. This work
was supported by the National Research Foundation
of Korea (NRF) grant funded by the Korea govern-
ment (MSIP) (NRF-2010-0027963) and Kwangwoon
University in 2014. AA and DB thank University Grant
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